Summary of Work: We are examining the relationship between the X ray crystal structures of DNA polymerases and their fidelity. To better understand the fidelity of base excision repair, this year we completed studies of the base substitution and frameshift fidelity of pol beta during synthesis to fill gaps of one, five, six, or >300 nucleotides. The data have implications for understanding the fidelity of synthesis during repair of a variety of DNA lesions resulting from endogenous and exogenous sources. We found evidence that the error specificity of pol beta depends on interactions between a specific arginine side chain at the active site and the DNA minor groove. These types of interactions, or the lack thereof, may generally explain previously ill-understood polymerase error specificity. We continued to study base-substitution mutator derivatives of Klenow DNA polymerase that result from substitution of highly conserved amino acid side chains located on the exposed surface of the polymerase cleft near the polymerase active site. Each has different error specificity, suggesting that the Arg- 668, Arg-682, Glu-710, Tyr-766 and Asn-845 side chains may contribute to polymerase fidelity in a variety of different ways. Finally, we studied the fidelity of copying triplet repeat sequences whose instability is associated with numerous degenerative human diseases. This study demonstates that DNA synthesis by polymerases essential for replication and repair generate deletions of discrete numbers of repeats during copying of triplet repeat sequences at rate much higher than for base substitution error rate. Deletions of many repeat units were generated during a single round of synthesis, demonstrating that multiple replication events are not necessary for large changes. Misaligned triplet repeat sequences were proofread but at a reduced efficiency compared to editing of single-base mismatches. Structure- function studies of this type probe several concepts for how DNA polymerases accurately copy DNA. It is our belief that they will improve our understanding of how the human genome is stably replicated and maintained, and how DNA adducts affect genome stability. - Antimutator, Base Selectivity, Base Excision Repair, DNA Polymerases, Mutator, Proofreading